Substituted 1,2,4-oxadiazoles as TRPA1 inhibitors

ABSTRACT

The present disclosure provides certain substituted 1,2,4-oxadiazole derivatives of the below formula (I) that are inhibitors of transient receptor potential ankyrin 1 (TRPA1), and are therefore useful for the treatment of diseases treatable by inhibition of TRPA1. Also provided are pharmaceutical compositions containing the same, and processes for preparing said compounds.

FIELD OF THE INVENTION

The present disclosure provides certain imidazo[4,5-d]pyridazinonylderivatives that are inhibitors of transient receptor potential ankyrin1 (TRPA1), and are therefore useful for the treatment of diseasestreatable by inhibition of TRPA1. Also provided are pharmaceuticalcompositions containing the same, and processes for preparing saidcompounds.

BACKGROUND OF THE INVENTION

Transient receptor potential channels (TRP channels) are a group ofvoltage-gated ion channels located mostly on the plasma membrane ofnumerous mammalian cell types. There are approximately 30 structurallyrelated TRP channels sorted into groups: TRPA, TRPC, TRPM, TRPML, TRPN,TRPP and TRPV. Transient receptor potential cation channel, subfamily A,member 1 (TRPA1), also known as transient receptor potential ankyrin 1,is the only member of the TRPA gene subfamily. Structurally, TRPAchannels are characterized by multiple N-terminal ankyrin repeats (˜14in the N-terminus of human TRPA1) that gives rise to the “A” for ankyrindesignation (Montell, 2005).

TRPA1 is highly expressed in the plasma membrane of sensory neurons inthe dorsal root and nodose ganglia that serve both skin and lung, aswell as in small intestine, colon, pancreas, skeletal muscle, heart,brain, bladder and lymphocytes (https://www.proteinatlas.org/) as wellas in human lung fibroblasts.

TRPA1 is best known as a sensor for environmental irritants giving riseto somatosensory modalities such as pain, cold and itch. TRPA1 isactivated by a number of reactive, electrophilic stimuli (e.g. allylisothiocyanate, reactive oxygen species), as well as non-reactivecompounds (e.g. icilin), implicated in cough associated with asthma,chronic pulmonary obstructive disease (COPD), idiopathic pulmonaryfibrosis (IPF) or post-viral cough or for chronic idiopathic cough aswell as cough in sensitive patients. (Song and Chang, 2015; Grace andBelvisi, 2011). TRPA1 inhibitors are useful in the treatment of IPF inwhich cough is highly prevalent because of the link between cough andlung injury, based on studies showing cough-induced elevation of TGF-β(Xie et al., 2009; Froese et al., 2016; Tschumperlin et al., 2003;Yamamoto et al., 2002; Ahamed et al., 2008). Acute lung injury as aresult of SARS-Cov-2 infection is mediated at least in part via reactiveoxygen species (ROS). ROS are a direct activator of TRPA1. Furthermore,desensitisation of TRPA1 via consumption of spicy foods has beenpostulated to regulate the Nrf2 pathway and reduce oxidative stress(Bousquet et al., 2020, Bousquet et al., 2021). TRPA1 inhibitorstherefore have the potential in the treatment of Covid-19/SARS-Cov-2induced lung injury. TRPA1 antagonists inhibit calcium signalingtriggered by cough triggers such as cigarette smoke extract (CSE)oxidative stress, inflammatory mediator release and downregulatedantioxidant gene expression (Lin et al., 2015; Wang et al., 2019). TRPA1antagonists are effective in studies of atopic dermatitis (Oh et al.,2013; Wilson et al., 2013), contact dermatitis (Liu et al., 2013),psoriasis-associated itch (Wilson et al., 2013) and IL-31-dependent itch(Cevikbas et al., 2014). A human TRPA1 gain-of-function has beenassociated with familial episodic pain syndrome (Kremeyer et al., 2010).A TRPA1 antagonist was effective in a behavioral model ofmigraine-related allodynia (Edelmayer et al., 2012). TRPA1 isselectively increased in trigeminal ganglia innervating injured teethwhen compared to TRPA1 expression in trigeminal ganglia innervatinghealthy teeth (Haas et al., 2011). Several anaesthetics are known to beTRPA1 agonists, including isoflurane (Matta et al., 2008) providingrationale for TRPA1 inhibitors for the relief of post-surgical pain.TRPA1 knockout mice and wild type mice treated with a TRPA1 antagonistshowed anxiolytic- and antidepressant-like phenotypes (de Moura et al.,2014). TRPA1 inhibitors are expected to have benefit in the treatment ofdiabetic neuropathy based on studies showing a mechanistic link ofinverse regulation between AMPK and TRPA1 (Hiyama et al., 2018; Koivistoand Pertovaara, 2013; Wang et al., 2018). TRPA1 knockout mice exhibitsmaller myocardial infarct sizes compared to wild type mice (Conklin etal., 2019). TRPA1 knockout and pharmacological intervention inhibitedTNBS-induced colitis in mice (Engel et al., 2011). In a mouse brainischaemia model, TRPA1 knock-out and TRPA1 antagonists reduce myelindamage (Hamilton et al., 2016). Urate crystals and joint inflammationare reduced in TRPA1 knockout mice in a monosodium urate mouse model ofgout (Moilanen et al., 2015). TRPA1 deletion in rats ameliorated jointinflammation and hyperalgesia in a rat model of acute gout flares(Trevisan et al., 2014). Activation of TRPA1 elicits an inflammatoryresponse in osteoarthritic chondrocytes (Nummenmaa et al., 2016). TRPA1inhibition and genetic deletion reduces inflammatory mediators inosteoarthritic mouse chondrocytes and murine cartilage (Nummenmaa etal., 2016). Finally, TRPA1 knockout mice exhibited improvements inweight bearing on the osteoarthritic limb in an MIA-evoked knee swellingmodel (Horvath et al., 2016). TRPA1 is differentially expressed in thebladder epithelium of rats (Du et al., 2007) and of patients withbladder outlet obstruction (Du et al., 2008). TRPA1 receptor modulationattenuates bladder overactivity in a rat model of spinal cord injury(Andrade et al., 2011) and intrathecal administration of TRPA1antagonists attenuate cyclophosphamide-induced cystitis in rats withhyper-reflexia micturition (Chen et al., 2016).

It is therefore desirable to provide potent TRPA1 inhibitors.

TRPA1 inhibitors of various structural classes are reviewed in S.Skerratt, Progress in Medicinal Chemistry, 2017, Volume 56, 81-115 andin D. Preti, G. Saponaro, A. Szallasi, Pharm. Pat. Anal. (2015) 4 (2),75-94, and in H. Chen, Transient receptor potential ankyrin 1 (TRPA1)antagonists: a patent review (2015-2019), Expert Opin Ther Pat., 2020.

WO2017/060488 discloses compounds that are antagonists of TRPA1, havingthe generalized structural formula

The TRPA1 activities of Examples 53, 72, 73, 86 and 90 therein aredisclosed having IC₅₀'s of less than 100 nM in a calcium flux assay.

L. Schenkel, et al., J. Med. Chem. 2016, 59, 2794-2809 disclosesquinazolinone-based TRPA1 antagonists including compounds of thegeneralized structural formula

of which compound 31, wherein R is OH, is disclosed as having anantagonistic TRPA1 activity IC₅₀ of 58 nM in a FLIPR assay and having anintrinsic clearance in human liver microsomes of <14 μL/min/kg.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses novel imidazo[4,5-d]pyridazinonylderivatives that are inhibitors of transient receptor potential ankyrin1 (TRPA1), possessing appropriate pharmacological and pharmacokineticproperties enabling their use as medicaments for the treatment ofconditions and/or diseases treatable by inhibition of TRPA1.

The compounds of the present invention may provide several advantages,such as enhanced potency, high metabolic and/or chemical stability, highselectivity, safety and tolerability, enhanced solubility, enhancedpermeability, desirable plasma protein binding, enhancedbioavailability, suitable pharmacokinetic profiles, and the possibilityto form stable salts.

The Compounds of the Invention

The present invention provides imidazo[4,5-d]pyridazinonyl derivativesthat are surprisingly potent inhibitors of TRPA1 (Assay A), furthercharacterised by

-   -   improved stability in human liver microsomes (Assay B)    -   improved stability in human hepatocytes (Assay C)

Compounds of the present invention differ structurally from examples 53,72, 73, 86 and 90 in WO2017/060488 and from example 31 in L. Schenkel,et al., J. Med. Chem. 2016, 59, 2794-2809, in that they contain asubstituted imidazo[4,5-d]pyridazinonyl core as well as substituentsadjacent to a secondary aliphatic alcohol. These structural differencesunexpectedly lead to a favourable combination of (i) inhibition ofTRPA1, (ii) stability in human liver microsomes, and (iii) stability inhuman hepatocytes.

Stability in human liver microsomes refers to the susceptibility ofcompounds to biotransformation in the context of selecting and/ordesigning drugs with favorable pharmacokinetic properties as a firstscreening step. The primary site of metabolism for many drugs is theliver. Human liver microsomes contain the cytochrome P450s (CYPs), andthus represent a model system for studying phase I drug metabolism invitro. Enhanced stability in human liver microsomes is associated withseveral advantages, including increased bioavailability and adequatehalf-life, which can enable lower and less frequent dosing of patients.Thus, enhanced stability in human liver microsomes is a favorablecharacteristic for compounds that are to be used for drugs. Therefore,compounds of the present invention in addition to being able to inhibitTRPA1 are expected to have a favorable in vivo clearance and thus thedesired duration of action in humans.

Stability in human hepatocytes refers to the susceptibility of compoundsto biotransformation in the context of selecting and/or designing drugswith favorable pharmacokinetic properties. The primary site ofmetabolism for many drugs is the liver. Human hepatocytes contain thecytochrome P450s (CYPs) and other drug metabolizing enzymes, and thusrepresent a model system for studying drug metabolism in vitro.(Importantly, in contrast to liver microsomes assay, the hepatocytesassay covers also phase II biotransformations as well as liver-specifictransporter-mediated processes, and therefore represents a more completesystem for drug metabolism studies). Enhanced stability in humanhepatocytes is associated with several advantages, including increasedbioavailability and adequate half-life, which can enable lower and lessfrequent dosing of patients. Thus, enhanced stability in humanhepatocytes is a favorable characteristic for compounds that are to beused for drugs.

The present invention provides novel compounds according to formula (I)

wherein

A is selected from the group consisting of phenyl, thiophenyl,benzothiophenyl and benzofuranyl, and wherein A is unsubstituted orsubstituted with one or two members of the group R¹ consisting ofhalogen and C₁₋₄-alkyl;

or A is

E is selected from the group consisting of

Another embodiment of the present invention relates to a compound offormula (I), wherein A is selected from the group consisting of phenyl,thiophenyl, benzothiophenyl and benzofuranyl, and wherein A isunsubstituted or substituted with one or two members of the group R¹consisting of Br, Cl, F and H₃C;

or A is

and substituent E is defined as in the preceding embodiment.

Another embodiment of the present invention relates to a compound offormula (I), wherein A is selected from the group consisting of phenyl,benzothiophenyl and benzofuranyl, and wherein A is unsubstituted orsubstituted with one or two members of the group R¹ consisting ofhalogen and C₁₋₄-alkyl;

or A is

and substituent E is defined as in any of the preceding embodiments.

Another embodiment of the present invention relates to a compound offormula (I), wherein A is selected from the group consisting of phenyl,benzothiophenyl and benzofuranyl, and wherein A is unsubstituted orsubstituted with one or two members of the group R¹ consisting of Br,Cl, F and H₃C;

or A is

and substituent E is defined as in any of the preceding embodiments.

Another embodiment of the present invention relates to a compound offormula (I), wherein A is selected from the group consisting of

and wherein A is unsubstituted or substituted with one or two members ofthe group R¹,

or

A is

and substituents E and R¹ are defined as in any of the precedingembodiments.

Another embodiment of the present invention relates to a compound offormula (I), wherein

A is selected from the group consisting of

and substituent E is defined as in any of the preceding embodiments.

Another embodiment of the present invention relates to a compound offormula (I), wherein E is

and substituent A is defined as in any of the preceding embodiments.

Another embodiment of the present invention relates to a compound offormula (I), wherein E is

and substituent A is defined as in any of the preceding embodiments.

Particularly preferred is the compound according to formula (I) selectedfrom the group consisting of

Used Terms and Definitions

Terms not specifically defined herein should be given the meanings thatwould be given to them by one of skill in the art in light of thedisclosure and the context. As used in the specification, however,unless specified to the contrary, the following terms have the meaningindicated and the following conventions are adhered to.

In the groups, radicals, or moieties defined below, the number of carbonatoms is often specified preceding the group, for example, C₁₋₆-alkylmeans an alkyl group or radical having 1 to 6 carbon atoms. In generalin groups like HO, H₂N, (O)S, (O)₂S, NC (cyano), HOOC, F₃C or the like,the skilled artisan can see the radical attachment point(s) to themolecule from the free valences of the group itself. For combined groupscomprising two or more subgroups, the last named subgroup is the radicalattachment point, for example, the substituent “aryl-C₁₋₃-alkyl” meansan aryl group which is bound to a C₁₋₃-alkyl-group, the latter of whichis bound to the core or to the group to which the substituent isattached.

In case a compound of the present invention is depicted in form of achemical name and as a formula in case of any discrepancy the formulashall prevail. An asterisk may be used in sub-formulas to indicate thebond which is connected to the core molecule as defined.

The numeration of the atoms of a substituent starts with the atom thatis closest to the core or to the group to which the substituent isattached.

For example, the term “3-carboxypropyl-group” represents the followingsubstituent:

wherein the carboxy group is attached to the third carbon atom of thepropyl group. The terms “1-methylpropyl-”, “2,2-dimethylpropyl-” or“cyclopropylmethyl-” group represent the following groups:

The asterisk may be used in sub-formulas to indicate the bond that isconnected to the core molecule as defined.

The term “C_(1-n)-alkyl”, wherein n is an integer selected from 2, 3, 4or 5, either alone or in combination with another radical denotes anacyclic, saturated, branched or linear hydrocarbon radical with 1 to n Catoms. For example the term C₁₋₅-alkyl embraces the radicals H₃C−,H₃C—CH₂—, H₃C—CH₂—CH₂—, H₃C—CH(CH₃)—, H₃C—CH₂—CH₂—CH₂—,H₃C—CH₂—CH(CH₃)—, H₃C—CH(CH₃)—CH₂—, H₃C—C(CH₃)₂—, H₃C—CH₂—CH₂—CH₂—CH₂—,H₃C—CH₂—CH₂—CH(CH₃)—, H₃C—CH₂—CH(CH₃)—CH₂—, H₃C—CH(CH₃)—CH₂—CH₂—,H₃C—CH₂—C(CH₃)₂—, H₃C—C(CH₃)₂—CH₂—, H₃C—CH(CH₃)—CH(CH₃)— andH₃C—CH₂—CH(CH₂CH₃)—.

The term “fluoro” added to an “alkyl”, “alkylene” or “cycloalkyl” group(saturated or unsaturated) means such a alkyl or cycloalkyl groupwherein one or more hydrogen atoms are replaced by a fluorine atom.Examples include, but are not limited to: H₂FC—, HF₂C— and F₃C—.

The term phenyl refers to the radical of the following ring

The term thiophenyl refers to the radical of the following ring

The term benzofuranyl refers to the radical of the following ring

The term benzothiophenyl refers to the radical of the following ring

The term imidazo[4,5-d]pyridazinonyl refers to the radical of thefollowing bicyclic cores

The term “substituted” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valence isnot exceeded, and that the substitution results in a stable compound.

Unless specifically indicated, throughout the specification and theappended claims, a given chemical formula or name shall encompasstautomers and all stereo, optical and geometrical isomers (e.g.enantiomers, diastereomers, E/Z isomers etc.) and racemates thereof aswell as mixtures in different proportions of the separate enantiomers,mixtures of diastereomers, or mixtures of any of the foregoing formswhere such isomers and enantiomers exist, as well as salts, includingpharmaceutically acceptable salts thereof and solvates thereof such asfor instance hydrates including solvates of the free compounds orsolvates of a salt of the compound.

In general, substantially pure stereoisomers can be obtained accordingto synthetic principles known to a person skilled in the field, e.g. byseparation of corresponding mixtures, by using stereochemically purestarting materials and/or by stereoselective synthesis. It is known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis, e.g. starting from optically activestarting materials and/or by using chiral reagents.

Enantiomerically pure compounds of this invention or intermediates maybe prepared via asymmetric synthesis, for example by preparation andsubsequent separation of appropriate diastereomeric compounds orintermediates which can be separated by known methods (e.g. bychromatographic separation or crystallization) and/or by using chiralreagents, such as chiral starting materials, chiral catalysts or chiralauxiliaries.

Further, it is known to the person skilled in the art how to prepareenantiomerically pure compounds from the corresponding racemic mixtures,such as by chromatographic separation of the corresponding racemicmixtures on chiral stationary phases; or by resolution of a racemicmixture using an appropriate resolving agent, e.g. by means ofdiastereomeric salt formation of the racemic compound with opticallyactive acids or bases, subsequent resolution of the salts and release ofthe desired compound from the salt; or by derivatization of thecorresponding racemic compounds with optically active chiral auxiliaryreagents, subsequent diastereomer separation and removal of the chiralauxiliary group; or by kinetic resolution of a racemate (e.g. byenzymatic resolution); by enantioselective crystallization from aconglomerate of enantiomorphous crystals under suitable conditions; orby (fractional) crystallization from a suitable solvent in the presenceof an optically active chiral auxiliary.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, and commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salt” refers to derivativesof the disclosed compounds wherein the parent compound forms a salt or acomplex with an acid or a base. Examples of acids forming apharmaceutically acceptable salt with a parent compound containing abasic moiety include mineral or organic acids such as benzenesulfonicacid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid,gentisic acid, hydrobromic acid, hydrochloric acid, maleic acid, malicacid, malonic acid, mandelic acid, methanesulfonic acid,4-methyl-benzenesulfonic acid, phosphoric acid, salicylic acid, succinicacid, sulfuric acid and tartaric acid.

Examples for cations and bases forming a pharmaceutically acceptablesalt with a parent compound containing an acidic moiety include Na⁺, K⁺,Ca²⁺, Mg²⁺, NH₄ ⁺, L-arginine, 2,2′-iminobisethanol, L-lysine,N-methyl-D-glucamine or tris(hydroxymethyl)-aminomethane. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha sufficient amount of the appropriate base or acid in water or in anorganic diluent like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile, or a mixture thereof.

Salts of other acids than those mentioned above which for example areuseful for purifying or isolating the compounds of the present invention(e.g. trifluoroacetate salts) also comprise a part of the presentinvention.

Biological Assays

Evaluation of TRPA1 Activity

Assay A: TRPA1 Assay

The activity of the compounds of the invention may be demonstrated usingthe following in vitro TRPA1 cell assay:

Method:

A human HEK293 cell line over-expressing the human TRPA1 ion channel(Perkin Elmer, Product No. AX-004-PCL) is used as a test system forcompound efficacy and potency. Compound activity is determined bymeasuring the effect of compounds on intracellular calcium concentrationinduced by AITC (Allylisothiocyanat) agonism in a FLIPRtetra system(Molecular Devices).

Cell Culture:

The cells are obtained as frozen cells in cryo-vials and stored untiluse at −150° C.

Cells are grown in culture medium (MEM/EBSS medium with 10% FCS and 0.4mg/ML Geneticin). It is important that density does not exceed 90%confluence. For sub-culturing cells are detached from flasks by Versene.At the day before the assay, cells are detached, washed twice withmedium (MEM/EBSS medium with 10% FCS) and 20000 cells in 20 μl/well areseeded to Poly D-Lysin biocoated 384-well plates (black, clear bottom,Cat. 356697) from Corning. Plates are incubated for 24 hours at 37°C./5% CO2 before use in the assay.

Compound Preparation

The test compounds are dissolved in 100% DMSO at a concentration of 10mM and in a first step diluted in DMSO to a concentration of 5 mM,followed by serial dilution steps in 100% DMSO. Dilution factor andnumber of dilution steps may vary according to needs. Typically 8different concentrations by 1:5 dilutions are prepared, furtherintermediate dilutions (1:20) of the substances are carried out withHBSS/HEPES buffer (1×HEPES, Cat. 14065 from Gibco, 20 mM HEPES, Cat.83264 from SIGMA, 0.1% BSA Cat. 11926 from Invitrogen, pH 7.4

FLIPR Assay:

At the assay day cells are washed 3× with assay puffer, 20 μL bufferremaining in the wells after washing. 10 μL Ca6 kit (Cat. R8191MolecularDevices) loading buffer in HBSS/HEPES is added to the cells andthe plates are incubated with lid for 120 minutes at 37°/5% CO2. 10 μLof compound or controls in HBSS/HEPES buffer/5% DMSO from theintermediate dilution plate are carefully added to the wells.Luminescence (indicating the calcium influx or release) is read on theFLIPRtetra device for 10 minutes to monitor the compound induced effects(e.g. agonism). Finally 10 μL of the agonist AITC 50 μM dissolved inHBSS/HEPES buffer/0.05% DMSO (final concentration 10 μM) is added to thewells followed by an additional read on the FLIPRtetra device for 10minutes. The area under the signal curve (AUC) after AITC addition isused for IC50/% inhibition calculations

Data Evaluation and Calculation:

Each assay microtiter plate contains wells with vehicle (1% DMSO)controls instead of compound as controls for AITC induced luminescence(100% CTL; high controls) and wells with vehicle controls without AITCas controls for non-specific changes in luminescence (0% CTL; lowcontrols).

The analysis of the data is performed by the calculation of the areaunder signal curve of the individual wells. Based on this values the %value for the measurement of each substance concentration is calculated(AUC(sample)−AUC(low))*100/(AUC(high)−AUC(low)) using MegaLab software(in house development). The IC50 values are calculated from the %control values using MegaLab software. Calculation:[y=(a−d)/(1+(x/c){circumflex over ( )}b)+d], a=low value, d=high value;x=conc M; c=IC50 M; b=hill; y=% ctrl

TABLE 1 Biological data for compounds of the invention as obtained inAssay A hTRPA1 IC₅₀ Example [nM] 1 22 2 47 3 147 4 158 5 60 6 96 7 204 841 9 9 10 13 11 19 12 24 13 9 14 19 15 47 16 79

TABLE 2 Biological data for prior art compounds (examples 53, 72, 73,86, 90 in WO2017/060488) as obtained in Assay A. Example in hTRPA1 IC₅₀WO2017/060488 [nM] 53 36 72 14 73 28 86 67 90 41

TABLE 3 Biological data for prior art compounds (example 31 in L.Schenkel, et al., J. Med. Chem. 2016, 59, 2794-2809) as obtained inAssay A. Example in Med. Chem. hTRPA1 IC₅₀ 2016, 59, 2794-2809 [nM] 3152

Evaluation of Microsomal Clearance

Assay B: Microsomal Clearance:

The metabolic degradation of the test compound is assayed at 37° C. withpooled liver microsomes. The final incubation volume of 100 μl per timepoint contains TRIS buffer pH 7.6 at RT (0.1 M), magnesium chloride (5mM), microsomal protein (1 mg/ml) and the test compound at a finalconcentration of 1 μM.

Following a short preincubation period at 37° C., the reactions areinitiated by addition of beta-nicotinamide adenine dinucleotidephosphate, reduced form (NADPH, 1 mM) and terminated by transferring analiquot into solvent after different time points (0, 5, 15, 30, 60 min).Additionally, the NADPH-independent degradation is monitored inincubations without NADPH, terminated at the last time point. The [%]remaining test compound after NADPH independent incubation is reflectedby the parameter c (control) (metabolic stability). The quenchedincubations are pelleted by centrifugation (10000 g, 5 min).

An aliquot of the supernatant is assayed by LC-MS/MS for the amount ofparent compound. The half-life (t½ INVITRO) is determined by the slopeof the semilogarithmic plot of the concentration-time profile.

The intrinsic clearance (CL_INTRINSIC) is calculated by considering theamount of protein in the incubation:CL _INTRINSIC [μl/min/mg protein]=(Ln 2/(half-life [min]*protein content[mg/ml]))*1000CL_INTRINSIC_INVIVO [ml/min/kg]=(CL_INTRINSIC [μL/min/mg protein]×MPPGL[mg protein/g liver]×liver factor [g/kg bodyweight])/1000Qh [%]=CL [ml/min/kg]/hepatic blood flow [ml/min/kg])

Hepatocellularity, human: 120×10e6 cells/g liver

Liver factor, human: 25.7 g/kg bodyweight

Blood flow, human: 21 ml/(min×kg)

TABLE 4 Biological data for compounds of the invention as obtained inAssay B Example human LM [% Qh] 1 <23 2 <23 3 <23 4 <23 5 <23 6 <23 7<23 8 <23 9 41 10 <23 11 30 12 31 13 36 14 <23 15 <23 16 <23

TABLE 5 Biological data for prior art compounds (examples 53, 72, 73,86, 90 in WO2017/060488) as obtained in Assay B. Example in human LMWO2017/060488 [% Qh] 53 <23 72 30 73 38 86 <23 90 39

TABLE 6 Biological data for prior art compounds (example 31 in L.Schenkel, et al., J. Med. Chem. 2016, 59, 2794-2809) as obtained inAssay B. Example in Med. Chem. human LM 2016, 59, 2794-2809 [% Qh] 31<23

Evaluation of Hepatocyte Clearance

Assay C: Hepatocyte Clearance

The metabolic degradation of the test compound is assayed in ahepatocyte suspension. Hepatocytes (cryopreserved) are incubated inDulbecco's modified eagle medium (supplemented with 3.5 μg glucagon/500mL, 2.5 mg insulin/500 mL and 3.75 mg/500 mL hydrocortison) containing5% species serum.

Following a 30 min preincubation in an incubator (37° C., 10% CO2) 5 μlof test compound solution (80 μM; from 2 mM in DMSO stock solutiondiluted 1:25 with medium) are added into 395 μl hepatocyte suspension(cell density in the range 0.25-5 Mio cells/mL depending on the species,typically 1 Mio cells/mL; final concentration of test compound 1 μM,final DMSO concentration 0.05%).

The cells are incubated for six hours (incubator, orbital shaker) andsamples (251) are taken at 0, 0.5, 1, 2, 4 and 6 hours. Samples aretransferred into acetonitrile and pelleted by centrifugation (5 min).The supernatant is transferred to a new 96-deepwell plate, evaporatedunder nitrogen and resuspended.

Decline of Parent Compound is Analyzed by HPLC-MS/MS

CLint is calculated as followsCL_INTRINSIC=Dose/AUC=(C0/CD)/(AUD+clast/k)×1000/60. C0: initialconcentration in the incubation [μM], CD: cell density of vital cells[10e6 cells/mL], AUD: area under the data [μM×h], clast: concentrationof last data point [μM], k: slope of the regression line for parentdecline [h−1].

The calculated in vitro hepatic intrinsic clearance can be scaled up tothe intrinsic in vivo hepatic Clearance and used to predict hepatic invivo blood clearance (CL) by the use of a liver model (well stirredmodel).CL_INTRINSIC_INVIVO [ml/min/kg]=(CL_INTRINSIC[μL/min/10e6cells]×hepatocellularity [10e6 cells/g liver]×liver factor[g/kg bodyweight])/1000CL [ml/min/kg]=CL_INTRINSIC_INVIVO [ml/min/kg]×hepatic blood flow[ml/min/kg]/(CL_INTRINSIC_INVIVO [ml/min/kg]+hepatic blood flow[ml/min/kg])Qh [%]=CL [ml/min/kg]/hepatic blood flow [ml/min/kg])

Hepatocellularity, human: 120×10e6 cells/g liver

Liver factor, human: 25.7 g/kg bodyweight

Blood flow, human: 21 ml/(min×kg)

TABLE 7 Biological data for compounds of the invention as obtained inAssay C human Hepatocytes Example [% Qh] 1 10 2 14 3 13 4 9 5 23 6 9 7 98 39 9 43 10 29 11 35 12 42 14 43 15 12 16 14

TABLE 8 Biological data for prior art compounds (examples 53, 72, 73,86, 90 in WO2017/060488) as obtained in Assay C. Example in humanHepatocytes WO2017/060488 [% Qh] 53 25 72 50 73 36 86 12 90 61

TABLE 9 Biological data for prior art compounds (example 31 in L.Schenkel, et al., J. Med. Chem. 2016, 59, 2794-2809) as obtained inAssay C. Example in Med. Chem. human Hepatocytes 2016, 59, 2794-2809 [%Qh] 31 73

Evaluation of Permeability

Caco-2 cells (1-2×10⁵ cells/1 cm² area) are seeded on filter inserts(Costar transwell polycarbonate or PET filters, 0.4 μm pore size) andcultured (DMEM) for 10 to 25 days. Compounds are dissolved inappropriate solvent (like DMSO, 1-20 mM stock solutions). Stocksolutions are diluted with HTP-4 buffer (128.13 mM NaCl, 5.36 mM KCl, 1mM MgSO₄, 1.8 mM CaCl₂, 4.17 mM NaHCO₃, 1.19 mM Na₂HPO₄×7H₂O, 0.41 mMNaH₂PO₄×H₂O, 15 mM HEPES, 20 mM glucose, 0.25% BSA, pH 7.2) to preparethe transport solutions (0.1-300 μM compound, final DMSO<=0.5%). Thetransport solution (TL) is applied to the apical or basolateral donorside for measuring A-B or B-A permeability (3 filter replicates),respectively. Samples are collected at the start and end of experimentfrom the donor and at various time intervals for up to 2 hours also fromthe receiver side for concentration measurement by HPLC-MS/MS orscintillation counting. Sampled receiver volumes are replaced with freshreceiver solution.

Evaluation of Plasma Protein Binding

This equilibrium dialysis (ED) technique is used to determine theapproximate in vitro fractional binding of test compounds to plasmaproteins. Dianorm Teflon dialysis cells (micro 0.2) are used. Each cellconsists of a donor and an acceptor chamber, separated by an ultrathinsemipermeable membrane with a 5 kDa molecular weight cutoff. Stocksolutions for each test compound are prepared in DMSO at 1 mM anddiluted to a final concentration of 1.0 μM. The subsequent dialysissolutions are prepared in pooled human or rat plasma (with NaEDTA) frommale and female donors. Aliquots of 200 μL dialysis buffer (100 mMpotassium phosphate, pH 7.4) are dispensed into the buffer chamber.Aliquots of 200 μL test compound dialysis solution are dispensed intothe plasma chambers. Incubation is carried out for 2 hours underrotation at 37° C.

At the end of the dialysis period, the dialysate is transferred intoreaction tubes. The tubes for the buffer fraction contain 0.2 mLACN/water (80/20). Aliquots of 25 μL of the plasma dialysate aretransferred into deep well plates and mixed with 25 μL ACN/water(80/20), 25 μL buffer, 25 μL calibration solution and 25 μL InternalStandard solution. Protein precipitation is done by adding 200 μL ACN.Aliquots of 50 μL of the buffer dialysate are transferred into deep wellplates and mixed with 25 μL blank plasma, 25 μL Internal Standardsolution and 200 μL ACN. Samples are measured on HPLC-MS/MS-Systems andevaluated with Analyst-Software. Percent bound is calculated with theformula: % bound=(plasma concentration−buffer concentration/plasma 30concentration)×100.

Evaluation of Solubility

Saturated solutions are prepared in well plates (format depends onrobot) by adding an appropriate volume of selected aqueous media(typically in the range of 0.25-1.5 ml) into each well which contains aknown quantity of solid drug substance (typically in the range 0.5-5.0mg). The wells are shaken or stirred for a predefined time period(typically in a range of 2-24 h) and than filtered using appropriatefilter membranes (typically PTFE-filters with 0.45 μm pore size). Filterabsorption is avoided by discarding the first few drops of filtrate. Theamount of dissolved drug substance is determined by UV spectroscopy. Inaddition the pH of the aqueous saturated solution is measured using aglass-electrode pH meter.

Evaluation of Pharmacokinetic Characteristics

The test compound is administered either intravenously or orally to therespective test species. Blood samples are taken at several time pointspost application of the test compound, anticoagulated and centrifuged.

The concentration of analytes—the administered compound and/ormetabolites—are quantified in the plasma samples. PK parameters arecalculated using non compartment methods. AUC and Cmax are normalized toa dose of 1 μmol/kg.

Evaluation of Metabolism in Human Hepatocytes In Vitro

The metabolic pathway of a test compound is investigated using primaryhuman hepatocytes in suspension. After recovery from cryopreservation,human hepatocytes are incubated in Dulbecco's modified eagle mediumcontaining 5% human serum and supplemented with 3.5 μg glucagon/500 ml,2.5 mg insulin/500 ml and 3.75 mg/500 ml hydrocortisone. Following a 30min preincubation in a cell culture incubator (37° C., 10% CO₂), testcompound solution is spiked into the hepatocyte suspension to obtain afinal cell density of 1.0*10⁶ to 4.0*10⁶ cells/ml (depending on themetabolic turnover rate of the compound observed with primary humanhepatocytes), a final test compound concentration of 10 μM, and a finalDMSO concentration of 0.05%.

The cells are incubated for six hours in a cell culture incubator on ahorizontal shaker, and samples are removed from the incubation after 0,0.5, 1, 2, 4 or 6 hours, depending on the metabolic turnover rate.Samples are quenched with acetonitrile and pelleted by centrifugation.The supernatant is transferred to a 96-deepwell plate, evaporated undernitrogen and resuspended prior to bioanalysis by liquidchromatography-high resolution mass spectrometry for identification ofputative metabolites.

The structures are assigned tentatively based onFourier-Transform-MS^(n) data. Metabolites are reported as percentage ofthe parent in human hepatocyte incubation with a threshold of ≥4%.

Method of Treatment

The present invention is directed to compounds of general formula 1which are useful in the prevention and/or treatment of a disease and/orcondition associated with or modulated by TRPA1 activity, including butnot limited to the treatment and/or prevention of fibrotic disease,inflammatory and immunoregulatory disorders, respiratory orgastrointestinal diseases or complaints, ophthalmic diseases,inflammatory diseases of the joints and inflammatory diseases of thenasopharynx, eyes, and skin and pain and neurological disorders. Saiddisorders, diseases and complaints include cough, idiopathic pulmonaryfibrosis, other pulmonary interstitial diseases and other fibrotic,asthma or allergic diseases, eosinophilic diseases, chronic obstructivepulmonary disease, as well as inflammatory and immunoregulatorydisorders, such as rheumatoid arthritis and atherosclerosis, as well aspain and neurological disorders, such as acute pain, surgical pain,chronic pain and depression and bladder disorders.

The compounds of general formula 1 are useful for the prevention and/ortreatment of:

(1) Cough such as chronic idiopathic cough or chronic refractory cough,cough associated with asthma, COPD, lung cancer, post-viral infectionand idiopathic pulmonary fibrosis and other pulmonary interstitialdiseases.

(2) Pulmonary fibrotic diseases such as pneumonitis or interstitialpneumonitis associated with collagenosis, e.g. lupus erythematodes,systemic scleroderma, rheumatoid arthritis, polymyositis anddermatomysitis, idiopathic interstitial pneumonias, such as pulmonarylung fibrosis (IPF), non-specific interstitial pneumonia, respiratorybronchiolitis associated interstitial lung disease, desquamativeinterstitial pneumonia, cryptogenic orgainizing pneumonia, acuteinterstitial pneumonia and lymphocytic interstitial pneumonia,lymangioleiomyomatosis, pulmonary alveolar proteinosis, Langerhan's cellhistiocytosis, pleural parenchymal fibroelastosis, interstitial lungdiseases of known cause, such as interstitial pneumonitis as a result ofoccupational exposures such as asbestosis, silicosis, miners lung (coaldust), farmers lung (hay and mould), Pidgeon fanciers lung (birds) orother occupational airbourne triggers such as metal dust ormycobacteria, or as a result of treatment such as radiation,methotrexate, amiodarone, nitrofurantoin or chemotherapeutics, or forgranulomatous disease, such as granulomatosis with polyangitis,Churg-Strauss syndrome, sarcoidosis, hypersensitivity pneumonitis, orinterstitial pneumonitis caused by different origins, e.g. aspiration,inhalation of toxic gases, vapors, bronchitis or pneumonitis orinterstitial pneumonitis caused by heart failure, X-rays, radiation,chemotherapy, M. boeck or sarcoidosis, granulomatosis, cystic fibrosisor mucoviscidosis, alpha-1-antitrypsin deficiency, acute lung injury asa result of Covid-19/SARS-Cov-2 infection or pulmonary fibrosissecondary to Covid-19/SARS-Cov-2 infection.

(3) Other fibrotic diseases such as hepatic bridging fibrosis, livercirrhosis, non-alcoholic steatohepatitis (NASH), atrial fibrosis,endomyocardial fibrosis, old myocardial infarction, glial scar, arterialstiffness, arthrofibrosis, Dupuytren's contracture, keloid,scleroderma/systemic sclerosis, mediastinal fibrosis, myelofibrosis,Peyronie's disease, nephrogenic systemic fibrosis, retroperitonealfibrosis, adhesive capsulitis.

(4) Inflammatory, auto-immune or allergic diseases and conditions suchas allergic or non-allergic rhinitis or sinusitis, chronic sinusitis orrhinitis, nasal polyposis, chronic rhinosinusitis, acute rhinosinusitis,asthma, pediatric asthma, allergic bronchitis, alveolitis, hyperreactiveairways, allergic conjunctivitis, bronchiectasis, adult respiratorydistress syndrome, bronchial and pulmonary edema, bronchitis orpneumonitis, eosinophilic cellulites (e.g., Well's syndrome),eosinophilic pneumonias (e.g., Loeffler's syndrome, chronic eosinophilicpneumonia), eosinophilic fasciitis (e. g., Shulman's syndrome),delayed-type hypersensitivity, non-allergic asthma; exercise inducedbronchoconstriction; chronic obstructive pulmonary disease (COPD), acutebronchitis, chronic bronchitis, cough, pulmonary emphysema; systemicanaphylaxis or hypersensitivity responses, drug allergies (e.g., topenicillin, cephalosporin), eosinophiliamyalgia syndrome due to theingestion of contaminated tryptophane, insect sting allergies;autoimmune diseases, such as rheumatoid arthritis, Graves' disease,Sjogren's syndrome psoriatic arthritis, multiple sclerosis, systemiclupus erythematosus, myasthenia gravis, immune thrombocytopenia (adultITP, neonatal thrombocytopenia, pediatric ITP), immune hemolytic anemia(auto-immune and drug induced), Evans syndrome (platelet and red cellimmune cytopaenias), Rh disease of the newborn, Goodpasture's syndrome(anti-GBM disease), Celiac, autoimmune cardio-myopathy juvenile onsetdiabetes; glomerulonephritis, autoimmune thyroiditis, Behcet's disease;graft rejection (e.g., in transplantation), including allograftrejection or graft versus-host disease; inflammatory bowel diseases,such as Crohn's disease and ulcerative colitis; spondyloarthropathies;scleroderma; psoriasis (including T-cell mediated psoriasis) andinflammatory dermatoses such as an dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria; vasculitis (e. g.,necrotizing, cutaneous, and hypersensitivity vasculitis); erythemanodosum; eosinophilic myositis, eosinophilic fasciitis, cancers withleukocyte infiltration of the skin or organs; ophthalmic diseases suchas age related macular degeneration, diabetic retinopathy and diabeticmacular edema, keratitis, eosinophilic keratitis, keratoconjunctivitis,vernal keratoconjunctivitis, scarring, anterior segment scarring,blepharitis, blepharoconjunctivitis, bullous disorders, cicatricialpemphigoid, conjunctival melanoma, papillary conjunctivitis, dry eye,episcleritis, glaucoma, gliosis, Granuloma annulare, Graves'ophthalmopathy, intraocular melanoma, Pinguecula, proliferativevitreoretinopathy, pterygia, scleritis, uveitis, acute gout flares, goutor osteoarthritis.

(5) Pain such as chronic idiopathic pain syndrome, neuropathic pain,dysesthesia, allodynia, migraine, dental pain and post-surgical pain.

(6) Depression, anxiousness, diabetic neuropathy and bladder disorderssuch as bladder outlet obstruction, overactive bladder, cystitis;myocardial reperfusion injury or brain ischaemia injury.

Accordingly, the present invention relates to a compound of generalformula 1 for use as a medicament.

Furthermore, the present invention relates to the use of a compound ofgeneral formula 1 for the treatment and/or prevention of a diseaseand/or condition associated with or modulated by TRPA1 activity.

Furthermore, the present invention relates to the use of a compound ofgeneral formula 1 for the treatment and/or prevention of fibroticdisease, inflammatory and immunoregulatory disorders, respiratory orgastrointestinal diseases or complaints, ophthalmic diseases,inflammatory diseases of the joints and inflammatory diseases of thenasopharynx, eyes, and skin, pain and neurological disorders. Saiddisorders, diseases and complaints include cough, idiopathic pulmonaryfibrosis, other pulmonary interstitial diseases and other fibrotic,asthma or allergic diseases, eosinophilic diseases, chronic obstructivepulmonary disease, as well as inflammatory and immunoregulatorydisorders, such as rheumatoid arthritis and atherosclerosis, as well aspain and neurological disorders, such as acute pain, surgical pain,chronic pain and depression and bladder disorders.

Furthermore, the present invention relates to the use of a compound ofgeneral formula 1 for the treatment and/or prevention of:

(1) Cough such as chronic idiopathic cough or chronic refractory cough,cough associated with asthma, COPD, lung cancer, post-viral infectionand idiopathic pulmonary fibrosis and other pulmonary interstitialdiseases.

(2) Pulmonary fibrotic diseases such as pneumonitis or interstitialpneumonitis associated with collagenosis, e.g. lupus erythematodes,systemic scleroderma, rheumatoid arthritis, polymyositis anddermatomysitis, idiopathic interstitial pneumonias, such as pulmonarylung fibrosis (IPF), non-specific interstitial pneumonia, respiratorybronchiolitis associated interstitial lung disease, desquamativeinterstitial pneumonia, cryptogenic orgainizing pneumonia, acuteinterstitial pneumonia and lymphocytic interstitial pneumonia,lymangioleiomyomatosis, pulmonary alveolar proteinosis, Langerhan's cellhistiocytosis, pleural parenchymal fibroelastosis, interstitial lungdiseases of known cause, such as interstitial pneumonitis as a result ofoccupational exposures such as asbestosis, silicosis, miners lung (coaldust), farmers lung (hay and mould), Pidgeon fanciers lung (birds) orother occupational airbourne triggers such as metal dust ormycobacteria, or as a result of treatment such as radiation,methotrexate, amiodarone, nitrofurantoin or chemotherapeutics, or forgranulomatous disease, such as granulomatosis with polyangitis,Churg-Strauss syndrome, sarcoidosis, hypersensitivity pneumonitis, orinterstitial pneumonitis caused by different origins, e.g. aspiration,inhalation of toxic gases, vapors, bronchitis or pneumonitis orinterstitial pneumonitis caused by heart failure, X-rays, radiation,chemotherapy, M. boeck or sarcoidosis, granulomatosis, cystic fibrosisor mucoviscidosis, alpha-1-antitrypsin deficiency, acute lung injury asa result of Covid-19/SARS-Cov-2 infection or pulmonary fibrosissecondary to Covid-19/SARS-Cov-2 infection.

(3) Other fibrotic diseases such as hepatic bridging fibrosis, livercirrhosis, non-alcoholic steatohepatitis (NASH), atrial fibrosis,endomyocardial fibrosis, old myocardial infarction, glial scar, arterialstiffness, arthrofibrosis, Dupuytren's contracture, keloid,scleroderma/systemic sclerosis, mediastinal fibrosis, myelofibrosis,Peyronie's disease, nephrogenic systemic fibrosis, retroperitonealfibrosis, adhesive capsulitis.

(4) Inflammatory, auto-immune or allergic diseases and conditions suchas allergic or non-allergic rhinitis or sinusitis, chronic sinusitis orrhinitis, nasal polyposis, chronic rhinosinusitis, acute rhinosinusitis,asthma, pediatric asthma, allergic bronchitis, alveolitis, hyperreactiveairways, allergic conjunctivitis, bronchiectasis, adult respiratorydistress syndrome, bronchial and pulmonary edema, bronchitis orpneumonitis, eosinophilic cellulites (e.g., Well's syndrome),eosinophilic pneumonias (e.g., Loeffler's syndrome, chronic eosinophilicpneumonia), eosinophilic fasciitis (e. g., Shulman's syndrome),delayed-type hypersensitivity, non-allergic asthma; exercise inducedbronchoconstriction; chronic obstructive pulmonary disease (COPD), acutebronchitis, chronic bronchitis, cough, pulmonary emphysema; systemicanaphylaxis or hypersensitivity responses, drug allergies (e.g., topenicillin, cephalosporin), eosinophiliamyalgia syndrome due to theingestion of contaminated tryptophane, insect sting allergies;autoimmune diseases, such as rheumatoid arthritis, Graves' disease,Sjogren's syndrome psoriatic arthritis, multiple sclerosis, systemiclupus erythematosus, myasthenia gravis, immune thrombocytopenia (adultITP, neonatal thrombocytopenia, pediatric ITP), immune hemolytic anemia(auto-immune and drug induced), Evans syndrome (platelet and red cellimmune cytopaenias), Rh disease of the newborn, Goodpasture's syndrome(anti-GBM disease), Celiac, autoimmune cardio-myopathy juvenile onsetdiabetes; glomerulonephritis, autoimmune thyroiditis, Behcet's disease;graft rejection (e.g., in transplantation), including allograftrejection or graft versus-host disease; inflammatory bowel diseases,such as Crohn's disease and ulcerative colitis; spondyloarthropathies;scleroderma; psoriasis (including T-cell mediated psoriasis) andinflammatory dermatoses such as an dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria; vasculitis (e. g.,necrotizing, cutaneous, and hypersensitivity vasculitis); erythemanodosum; eosinophilic myositis, eosinophilic fasciitis, cancers withleukocyte infiltration of the skin or organs; ophthalmic diseases suchas age related macular degeneration, diabetic retinopathy and diabeticmacular edema, keratitis, eosinophilic keratitis, keratoconjunctivitis,vernal keratoconjunctivitis, scarring, anterior segment scarring,blepharitis, blepharoconjunctivitis, bullous disorders, cicatricialpemphigoid, conjunctival melanoma, papillary conjunctivitis, dry eye,episcleritis, glaucoma, gliosis, Granuloma annulare, Graves'ophthalmopathy, intraocular melanoma, Pinguecula, proliferativevitreoretinopathy, pterygia, scleritis, uveitis, acute gout flares, goutor osteoarthritis.

(5) Pain such as chronic idiopathic pain syndrome, neuropathic pain,dysesthesia, allodynia, migraine, dental pain and post-surgical pain.

(6) Depression, anxiousness, diabetic neuropathy and bladder disorderssuch as bladder outlet obstruction, overactive bladder, cystitis;myocardial reperfusion injury or brain ischaemia injury.

In a further aspect the present invention relates to a compound ofgeneral formula 1 for use in the treatment and/or prevention of abovementioned diseases and conditions.

In a further aspect the present invention relates to the use of acompound of general formula 1 for the preparation of a medicament forthe treatment and/or prevention of above mentioned diseases andconditions.

In a further aspect of the present invention the present inventionrelates to methods for the treatment or prevention of above mentioneddiseases and conditions, which method comprises the administration of aneffective amount of a compound of general formula 1 to a human being.

Combination Therapy

The compounds of the invention may further be combined with one or more,preferably one additional therapeutic agent. According to one embodimentthe additional therapeutic agent is selected from the group oftherapeutic agents useful in the treatment of diseases or conditionsdescribed hereinbefore, in particular associated with fibrotic diseases,inflammatory and immunoregulatory disorders, respiratory orgastrointestinal diseases or complaints, inflammatory diseases of thejoints or of the nasopharynx, eyes, and skin or conditions such as forexample cough, idiopathic pulmonary fibrosis, other pulmonaryinterstitial diseases, asthma or allergic diseases, eosinophilicdiseases, chronic obstructive pulmonary disease, atopic dermatitis aswell as autoimmune pathologies, such as rheumatoid arthritis andatherosclerosis, or therapeutic agents useful for the treatment ofophthalmic diseases, pain and depression.

Additional therapeutic agents that are suitable for such combinationsinclude in particular those, which, for example, potentiate thetherapeutic effect of one or more active substances with respect to oneof the indications mentioned and/or allow the dosage of one or moreactive substances to be reduced.

Therefore, a compound of the invention may be combined with one or moreadditional therapeutic agents selected from the group consisting ofantifibrotic agents, anti-tussive agents, anti-inflammatory agents,anti-atopic dermatitis agents, analgesics, anti-convulsants,anxiolytics, sedatives, skeletal muscle relaxants or anti-depressants.

Antifibrotic agents are for example nintedanib, pirfenidone,phosphodiesterase-IV (PDE4) inhibitors such as roflumilast, autotaxininhibitors such as GLPG-1690 or BBT-877; connective tissue growth factor(CTGF) blocking antibodies such as Pamrevlumab; B-cell activating factorreceptor (BAFF-R) blocking antibodies such as Lanalumab; alpha-V/beta-6blocking inhibitors such as BG-00011/STX-100, recombinant pentraxin-2(PTX-2) such as PRM-151; c-Jun N-terminal kinase (JNK) inhibitors suchas CC-90001; galectin-3 inhibitors such as TD-139; G-protein coupledreceptor 84 (GPR84) inhibitors such as GLPG1205; G-protein coupledreceptor 84/G-protein coupled receptor 40 dual inhibitors such asPBI-4050; Rho Associated Coiled-Coil Containing Protein Kinase 2 (ROCK2)inhibitors such as KD-025; heat shock protein 47 (HSP47) smallinterfering RNA such as BMS986263/ND-L02-s0201; Wnt pathway inhibitorsuch as SM-04646; LD4/PDE3/4 inhibitors such as Tipelukast; recombinantimmuno-modulatory domains of histidyl tRNA synthetase (HARS) such asATYR-1923; prostaglandin synthase inhibitors such as ZL-2102/SAR-191801;15-hydroxy-eicosapentaenoic acid (15-HEPE e.g. DS-102); Lysyl OxidaseLike 2 (LOXL2) inhibitors such as PAT-1251, PXS-5382/PXS-5338;phosphoinositide 3-kinases (PI3K)/mammalian target of rapamycin (mTOR)dual inhibitors such as HEC68498; calpain inhibitors such as BLD-2660;mitogen-activated protein kinase kinase kinase (MAP3K19) inhibitors suchas MG-S-2525; chitinase inhibitors such as OATD-01; mitogen-activatedprotein kinase-activated protein kinase 2 (MAPKAPK2) inhibitors such asMMI-0100; transforming growth factor beta 1 (TGF-beta1) smallinterfering RNA such as TRK250/BNC-1021; or lysophosphatidic acidreceptor antagonists such as BMS986278.

Anti-tussive agents are, for example, purinoceptor 3 (P2X3) receptorantagonists such as gefapixant, S-600918, BAY-1817080, or BLU-5937;neurokinin 1 (NK-1) receptor antagonist such as Orvepitant, Aprepitant;nicotinic acetylcholine receptor alpha 7 subunit stimulator such asATA-101/bradanicline; codeine, gabapentin, pregablin, or azithromycin.Anti-inflammatory agents are, for example, corticosteroids such asprednisolone or dexamethasone; cyclo-oxygenase-2 (COX2) inhibitors suchas celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib orlumiracoxib; prostaglandin E2 antagonists; leukotriene B4 antagonists;leukotriene D4 antagonists such as monteleukast; 5-lipoxygenaseinhibitors; or other nonsteroidal anti-inflammatory agents (NSAIDs) suchas aspirin, diclofenac, diflunisal, etodolac, ibuprofen or indomethacin.

Anti-atopic dermatitis agents are, for example, cyclosporin,methotrexate, mycophenolate mofetil, azathioprine, phosphodiesteraseinhibitors (e.g. apremilast, crisaborole), Janus Associated Kinase (JAK)inhibitors (e.g. tofacitinib), neutralizing antibodies against IL-4/IL13(e.g. dupilamab), IL-13 (e.g. lebrikizumab, tralokinumab) and IL-31(nemolizumab). Analgesics are, for example, of the opioid type, such asmorphine, oxymorphine, levopanol, oxycodon, propoxyphene, nalmefene,fentanyl, hydrocondon, hydromorphone, meripidine, methadone, nalorphine,naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine,pentazocine; or of the non-opioid type, such as acetophenamine.

Anti-depressants are, for example, tricyclic anti-depressants such asamitriptyline, clomipramine, despramine, doxepin, desipramine,imipramine, nortriptyline; selective serotonin reuptake inhibitoranti-depressants (SSRIs) such as fluoxetine, paroxetine, sertraline,citalopram, escitalopram; norepinephrine reuptake inhibitoranti-depressants (SNRIs) such as maprotiline, lofepramine, mirtazapine,oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion,hydroxybuproprion, nomifensine, viloxazine; dualserotonin-norepinephrine reuptake inhibitor anti-depressants (SNRIs)such as duloxetine, venlafaxine, desvenlafaxine, levomilnacipran;atypical antidepressants such as trazodone, mirtazapine, vortioxetine,vilazodone, bupropion; or monoamine oxidase inhibitor anti-depressants(MAOIs) such as tranylcypromine, phenelzine, or isocarboxazid.

Anxiolytics are, for example, benzodiazepines such as alprazolam,bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam,flurazepam, lorazepam, oxazepam, temazepam, triazolam, or tofisopam; orthey are nonbenzodiazepine hypnotics such as eszopiclone, zaleplon,zolpidem, or zopiclone; or they are carbamates e.g. meprobamate,carisoprodol, tybamate, or lorbamate; or they are antihistamines such ashydroxyzine, chlorpheniramine or diphenhydramine.

Sedatives are, for example, barbiturate sedatives, such as amobarbital,aprobarbital, butabarbital, butabital, mephobarbital, metharbital,methohexital, pentobarbital, secobarbital, talbutal, theamylal, orthiopental; or they are non-barbiturate sedatives such as glutethimide,meprobamate, methaqualone or dichloalphenazone.

Skeletal muscle relaxants are, for example, baclofen, meprobamate,carisoprodol, cyclobenzaprine, metaxalone, methocarbamol, tizanidine,chlorzoxazone or orphenadrine. Other suitable combination partners areinhibitors of Acetylcholinesterase inhibitors such as donepezil; 5-HT-3anatgonists such as ondansetron; metabotropic glutamate receptorantagonists; antiarrhythmics such as mexiletine or phenytoin; or NMDAreceptor antagonists. Further suitable combination partners areincontinence medications, for example, anticholinergics such asoxybutynin, tolterodine, darifenacin, fesoterodine, solifenacin ortrospium; or they are bladder muscle relaxants such as mirabegron; orthey are alpha blockers such as tamsulosin, alfuzosin, silodosin,doxazosin or terazosin.

The dosage for the combination partners mentioned above is usually ⅕ ofthe lowest dose normally recommended up to 1/1 of the normallyrecommended dose.

Therefore, in another aspect, this invention relates to the use of acompound according to the invention in combination with one or moreadditional therapeutic agents described hereinbefore and hereinafter forthe treatment of diseases or conditions which may be affected or whichare mediated by TRPA1, in particular diseases or conditions as describedhereinbefore and hereinafter.

In a further aspect this invention relates to a method for treating adisease or condition which can be influenced by the inhibition of TRPA1in a patient that includes the step of administering to the patient inneed of such treatment a therapeutically effective amount of a compoundof formula (I) or a pharmaceutically acceptable salt thereof incombination with a therapeutically effective amount of one or moreadditional therapeutic agents.

In a further aspect this invention relates to the use of a compound offormula (I) or a pharmaceutically acceptable salt thereof in combinationwith one or more additional therapeutic agents for the treatment ofdiseases or conditions which can be influenced by the inhibition ofTRPA1 in a patient in need thereof.

In yet another aspect the present invention relates to a method for thetreatment of a disease or condition mediated by TRPA1 activity in apatient that includes the step of administering to the patient,preferably a human, in need of such treatment a therapeuticallyeffective amount of a compound of the present invention in combinationwith a therapeutically effective amount of one or more additionaltherapeutic agents described in hereinbefore and hereinafter.

The use of the compound according to the invention in combination withthe additional therapeutic agent may take place simultaneously or atstaggered times.

The compound according to the invention and the one or more additionaltherapeutic agents may both be present together in one formulation, forexample a tablet or capsule, or separately in two identical or differentformulations, for example as a so-called kit-of-parts.

Consequently, in another aspect, this invention relates to apharmaceutical composition that comprises a compound according to theinvention and one or more additional therapeutic agents describedhereinbefore and hereinafter, optionally together with one or more inertcarriers and/or diluents.

In yet another aspect the present invention relates to the use of acompound according to the invention in a cough-measuring device.

Other features and advantages of the present invention will becomeapparent from the following more detailed examples which illustrate, byway of example, the principles of the invention.

Preparation

The compounds according to the present invention and their intermediatesmay be obtained using methods of synthesis which are known to the oneskilled in the art and described in the literature of organic synthesis.Preferably, the compounds are obtained in analogous fashion to themethods of preparation explained more fully hereinafter, in particularas described in the experimental section. In some cases, the order incarrying out the reaction steps may be varied. Variants of the reactionmethods that are known to the one skilled in the art but not describedin detail here may also be used.

The general processes for preparing the compounds according to theinvention will become apparent to the one skilled in the art studyingthe following schemes. Any functional groups in the starting materialsor intermediates may be protected using conventional protecting groups.These protecting groups may be cleaved again at a suitable stage withinthe reaction sequence using methods familiar to the one skilled in theart.

The compounds according to the invention are prepared by the methods ofsynthesis described hereinafter in which the substituents of the generalformulae have the meanings given herein before. These methods areintended as an illustration of the invention without restricting itssubject matter and the scope of the compounds claimed to these examples.Where the preparation of starting compounds is not described, they arecommercially obtainable or may be prepared analogously to knowncompounds or methods described herein. Substances described in theliterature are prepared according to the published methods of synthesis.Abbreviations are as defined in the Examples section.

In scheme 1, compounds of formula I and II can be synthesized viaN-alkylation of the appropriate heteroaromatic intermediates (A) or (B)with chloromethylen-oxadiazoles (D) in presence of a base such aspotassium carbonate. Alternatively, carboxylic acid (C) can beactivated, e.g. with CDI, and condensed with dihydroxypropanimidamides(E) to yield compounds of type II.

In scheme 2, alpha-cyano ketones of substructure (F) are reduced in anenantioselective fashion by using appropriate catalytic systems using atransition metal complex (of e.g. Ru or Ir) in combination with a chiralligand (e.g.[(1S,2S)-(−)-2-amino-1,2-diphenylethyl](4-toluenesulfonyl)amido) and ahydrogen source such as formic acid triethylamine complex.

Hydroxylamine is added to the corresponding cyano-alcohols (G) toprovide the dihydroxypropanimidamides (E). Ring-closure tochloromethylen-oxadiazoles (D) can be achieved by stirring the reactionmixture together with chloro acetyl chloride in presence of a base suchas DIPEA.

In scheme 3, 4,5-dichloro-2H-pyridazin-3-one can be N-alkylated with anappropriate protecting group such as benzyloxymethyl chloride (Bom-Cl)in presence of a base such as DBU. Subsequently, substitution of the4-Cl with nitrite and the 5-Cl position of (H) with hydroxide providesintermediate (J), which can be further reacted with ammonia to give (K).Reduction of the nitro group using an appropriate catalyst such as PtO₂under a hydrogen atmosphere yields the di-amine (L), which can befurther reacted with carbonyl diimidazole (CDI) to give (M). Finally,di-alkylation with a methylating reagent such as methyl iodide inpresence of a base such as potassium carbonate, and cleavage of theprotecting group gives intermediate (A).

In Scheme 4, imidazole (O) can be alkylated with an appropriatemethylating reagent such as methyl iodide (Mel) in presence of a basesuch as potassium carbonate. Intermediate (P) can be reacted to aldehyde(R) via reduction, with e.g. di-iso butyl aluminium hydride (DIBAL-H),and subsequent oxidation, e.g. with manganese (IV) oxide. Condensationof (R) with hydrazine under acidic conditions gives (S), which can befurther reduced to intermediate (B). Finally, alkylation of (B) withtert-butyl bromoacetate in presence of a base such as potassiumcarbonate provides ester (T), which can be hydrolyzed under acidconditions to give (C).

EXAMPLES

Preparation

The compounds according to the invention and their intermediates may beobtained using methods of synthesis which are known to the one skilledin the art and described in the literature of organic synthesis forexample using methods described in “Comprehensive OrganicTransformations”, 2nd Edition, Richard C. Larock, John Wiley & Sons,2010, and “March's Advanced Organic Chemistry”, 7th Edition, Michael B.Smith, John Wiley & Sons, 2013. Preferably the compounds are obtainedanalogously to the methods of preparation explained more fullyhereinafter, in particular as described in the experimental section. Insome cases the sequence adopted in carrying out the reaction schemes maybe varied. Variants of these reactions that are known to the skilledartisan but are not described in detail herein may also be used. Thegeneral processes for preparing the compounds according to the inventionwill become apparent to the skilled man on studying the schemes thatfollow. Starting compounds are commercially available or may be preparedby methods that are described in the literature or herein, or may beprepared in an analogous or similar manner. Before the reaction iscarried out, any corresponding functional groups in the startingcompounds may be protected using conventional protecting groups. Theseprotecting groups may be cleaved again at a suitable stage within thereaction sequence using methods familiar to the skilled man anddescribed in the literature for example in “Protecting Groups”, 3rdEdition, Philip J. Kocienski, Thieme, 2005, and “Protective Groups inOrganic Synthesis”, 4th Edition, Peter G. M. Wuts, Theodora W. Greene,John Wiley & Sons, 2006. The terms “ambient temperature” and “roomtemperature” are used interchangeably and designate a temperature ofabout 20° C., e.g. between 19 and 24° C.

Abbreviations: ACN acetonitrile Aq. aqueous Bom Benzyloxymethyl ° C.Degree celsius CDI Carbonyl diimidazole CyH/CH cyclohexane conc.concentrated DBU 1,8-diazabicycloundec-7-ene DCM dichloro methane DCE1,2-Dichloroethane DIPEA N,N-diisopropylethylamine DMAN,N-dimethylacetamide DMF N,N-dimethylformamide DMSO dimethyl sulfoxideESI-MS Electrospray ionisation mass spectrometry EtOAc ethyl acetateEtOH ethanol ex example eq equivalent FA formic acid h hour HATU1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium3-oxid hexafluorophosphate HCl Hydrochloric acid HPLC High performanceliquid chromatography Int. intermediate K₂CO₃ potassium carbonateK(OtBu) Potassium tert. butoxide L liter M molar MeOH methanol MgSO₄magnesium sulphate min minute mL milliliter MTBE tert-butylmethyletherNaOEt Sodium ethanolate NH₃ ammonia NMP N-methyl-2-pyrrolidon PE Petrolether RT room temperature (about 20° C.) sat. saturated TBTUBenzotriazolyl tetramethyluronium tetrafluoroborate TEA triethylamineTFA trifluoroacetic acid THF Tetrahydrofuran TMS-Cl Trimethylsilylchloride

Preparation of Intermediates

Intermediate I

Intermediate I.1 (General Route)

(3S)-3-(4-chlorophenyl)-3-hydroxypropanenitrile

10.0 g (55.7 mmol) 4-Chlorobenzoylacetonitrile are added to 100 mL ACNunder inert atmosphere. 142 mg (0.23 mmol)Chloro([(1S,2S)-2-amino-1,2-diphenylethyl](4-toluenesulfonyl)amido)(mesitylene)ruthenium(II) (CAS 174813-81-1) are added, followed by dropwise addition of 8.30mL (19.8 mmol) formic acid triethylamine complex (5:2). After stirringat RT for 3 h, the solvent is removed in vacuo. To the remaining crudemixture is added water and this mixture is extracted two times withEtOAc. The organic layers are combined, dried over MgSO₄, filtered, andthe solvent is removed in vacuo to provide intermediate I.1.

C₉H₈ClNO (M=181.6 g/mol)

ESI-MS: 226 [M+HCOO]⁻

R_(t) (HPLC): 0.81 min (method B)

The following compounds are prepared using procedures analogous to thosedescribed for intermediate I.1 using appropriate starting materials. Asis appreciated by those skilled in the art, these analogous examples mayinvolve variations in general reaction conditions.

HPLC retention time [min] (method), or 1H NMR (300 MHz, DMSO-d6) Int.Starting materials Structure ESI-MS δ ppm I.2

187 [M*]⁺ 0.41 (A) I.3

184 [M + Na]⁺ 0.76 (B) I.4

188 [M + Na]⁺ 0.72 (B) I.5

214 [M + Na]⁺ 0.64 (B) 1.6

248/250 [M + Na]⁺ 0.43 (A) 1.7 IV.1

266 [M + HCOO]⁻ 3.03 (L) 1.8

— δ 7.98-7.91 (m, 1 H), 7.82-7.77 (m, 1 H), 7.37-7.31 (m, 3 H), 6.56 (d,J = 5.0 Hz, 1 H), 5.28-5.20 (m, 1 H), 3.14-2.94 (m, 2 H) 1.9 IV.2

266 [M + HCOO]⁻ 3.12 (L) 1.10 IV.3

— δ 7.63-7.56 (m, 1 H), 7.46 (dd, J = 8.9, 2.7 Hz, 1 H), 7.14 (td, J =9.2, 2.7 Hz, 1 H), 6.88 (s, 1 H), 6.41 (d, J = 5.5 Hz, 1 H), 5.10-5.01(m, 1 H), 3.16-2.98 (m, 2 H)

Intermediate II

Intermediate II.1 (General Route)

(3S)-3-(4-chlorophenyl)-N,3-dihydroxypropanimidamidl

To 9.82 g (54.1 mmol) (3S)-3-(4-chlorophenyl)-3-hydroxypropanenitrile(intermediate 1.1) in 100 mL MeOH are added 8.00 mL (136 mmol)hydroxylamine (50% in water) and the mixture is stirred at 75° C. for1.5 h. After cooling to RT, all volatiles are removed in vacuo to yieldthe crude product, which is used without further purification.

C₉H₁₁ClN₂O₂ (M=214.6 g/mol)

ESI-MS: 215 [M+H]⁺

R_(t) (HPLC): 0.60 min (method B)

The following compounds are prepared using procedures analogous to thosedescribed for intermediate I1.1 using appropriate starting materials. Asis appreciated by those skilled in the art, these analogous examples mayinvolve variations in general reaction conditions.

HPLC retention time Starting [min] Int. materials Structure ESI-MS(method) II.2 I.2

221 [M + H]⁺ 0.27 (A) II.3 I.3

195 [M + H]⁺ 0.57 (B) II.4 I.4

199 [M + H]⁺ 0.43 (B) II.5 I.5

225 [M + H]⁺ 0.24 (B) II.6 1.6

259/261 [M + H]⁺ 0.66 (B) II.7 I.7

255 [M + H]⁺ 2.07 (L) II.8 I.8

237 [M + H]⁺ 1.93 (L) II.9 I.9

255 [M + H]⁺ 2.18 (L) II.10  I.10

239 [M + H]⁺ 1.90 (L)

Intermediate III

Intermediate III.1 (General Route)

(1S)-2-[5-(chloromethyl)-1,2,4-oxadiazol-3-yl]-1-(4-chlorophenyl)ethan-1-ol

To 11.2 g (52.4 mmol) of intermediate 11.1 in 55 mL NMP are added 10.0mL (57.8 mmol) DIPEA. The mixture is cooled to 0° C. before 4.60 mL(57.7 mmol) chloroacetyl chloride dissolved in 5 mL NMP are slowly addedand the mixture is stirred at 0° C. for 45 min. The mixture is thenheated up to 95° C. and stirring is continued for 4 h. After coolingdown to RT, 200 mL water are added and the resulting mixture isextracted three times with EtOAc.

The organic layers are combined, dried over MgSO₄, filtered and thesolvent is removed in vacuo. The residue is purified by columnchromatography (silica gel; PE/EtOAc, 7/3).

C₁₁H₁₀Cl₂N₂O₂ (M=273.1 g/mol)

ESI-MS: 271 [M−H]⁻

R_(t) (HPLC): 0.93 min (method B)

The following compounds are prepared using procedures analogous to thosedescribed for intermediate 111.1 using appropriate starting materials.As is appreciated by those skilled in the art, these analogous examplesmay involve variations in general reaction conditions.

HPLC retention Starting time [min] Int. materials Structure ESI-MS(method) III.2 II.2

301 [M + Na]⁺ 0.90 (B) III.3 II.3

251 [M − H]⁻ 0.92 (C) III.4 II.4

255 [M − H]⁻ 0.86 (B) III.5 II.5

281 [M − H]⁻ 0.82 (G) III.6 II.7

311 [M − H]⁻ 6.02 (M) III.7 II.8

295 [M + H]⁺ 5.88 (M) III.8 II.9

311 [M − H]⁻ 6.12 (M) III.9  II.10

295 [M − H]⁻ 5.67 (M)

Intermediate IV

Intermediate IV.1 (General Route)

3-(6-fluoro-1-benzothiophen-2-yl)-3-oxopropanenitrile

To 0.63 g (3.00 mmol) methyl 6-fluoro-1-benzothiophene-2-carboxylate in9.0 mL dry toluene and 0.78 mL dry ACN are added 0.36 g (9.00 mmol) ofNaH (60% in oil) under inert atmosphere at RT. The mixture is heated toreflux and stirred for 16 h, cooled to room temperature, poured onice/water (30 mL), and treated with 2M HCl to reach pH=1. EtOAc (20 mL)is added and the phases are separated. The aqueous phase is extractedonce more with EtOAc (20 mL), the combined organic phases are washedwith brine (20 mL), and the solvent is removed under reduced pressure.The crude product is purified by silica gel column chromatography usinga gradient of EtOAc/hexane (30% to 40%).

C₁₁H₆FNOS (M=219.23 g/mol)

ESI-MS: 218 [M−H]⁻

R_(t) (HPLC): 3.31 min (L)

The following compounds are prepared using procedures analogous to thosedescribed for intermediate IV.1 using appropriate starting materials. Asis appreciated by those skilled in the art, these analogous examples mayinvolve variations in general reaction conditions.

HPLC retention Starting time [min] Int. materials Structure ESI-MS(method) IV.2

218 [M − H]⁻ 3.33 (L) IV.3

202 [M − H]⁻ 3.08 (L)

Intermediate V

Intermediate V.1

2-[(benzyloxy)methyl]-4,5-dichloro-2,3-dihydropyridazin-3-on

To a stirred mixture of 300 g (1.82 mol) 4,5-dichloro-2H-pyridazin-3-onein 2 L DMF is added at 0° C. 262 g (1.72 mol)1,8-diazabicycloundec-7-ene followed by 370 g (2.37 mol)chloromethoxymethyl-benzene. The reaction mixture is allowed to warm toambient temperature and stirred for a total of 16 h. The mixture isdiluted with water and extracted with ethyl acetate (3×). The combinedorganic extracts are dried over Na₂SO₄ and concentrated.

The residue is purified by column chromatography (SiO₂, EtOAc/PE) toyield the desired product.

C₁₂H₁₁N₃O₅ (M=285.1 g/mol)

ESI-MS: 285/287 [M+H]⁺

R_(t) (HPLC): 2.2 min (K)

Intermediate V.2

2-[(benzyloxy)methyl]-4-hydroxy-5-nitro-2,3-dihydropyridazin-3-one

To 85.0 g (298 mmol) of intermediate V.1 in 700 mL DMF and 300 mL wateris added 82.3 g (1.19 mol) sodium nitrite. The reaction mixture isstirred at 85° C. for 24 h before it is cooled to ambient temperature,acidified with aqueous hydrochloric acid solution (5 N) and adjusted topH4. The mixture is filtered and the collected solid dried under vacuumto yield the desired product.

C₁₂H₁₁N₃O₅ (M=277.2 g/mol)

ESI-MS: 278 [M+H]⁺

R_(t) (HPLC): 1.71 min (method K)

Intermediate V.3

4-Amino-2-[(benzyloxy)methyl]-5-nitro-2,3-dihydropyridazin-3-one

To 50.0 g (180 mmol) of intermediate V.2 is added 3.75 L of a saturatedsolution of ammonia in MeOH. The reaction mixture is heated to 130° C.in an autoclave reactor overnight. After cooling to ambient temperature,the mixture is further cooled to 0° C. The precipitated solid iscollected by filtration and purified by column chromatography (SiO₂,DCM/MeOH gradient) to yield the desired product.

C₁₂H₁₂N₄O₄ (M=276.2 g/mol)

ESI-MS: 275 [M−H]⁻

R_(t) (HPLC): 1.76 min (method J)

Intermediate V.4

4,5-Diamino-2-[(benzyloxy)methyl]-2,3-dihydropyridazin-3-one

To a stirred mixture of 30.0 g (109 mmol) intermediate V.3 in 3650 mLEtOH/EtOAc (4:1) is added 2.47 g (10.9 mmol) platinum(IV) oxide. Themixture is degassed by passing Ar through the solvent for 15 min. Ahydrogen atmosphere (1 atm) is applied and the reaction mixture isstirred for 16 h. After removing the hydrogen atmosphere, the mixture isfiltered through a pad of celite and the filtrate is concentrated toyield the desired compound.

C₁₂H₁₄N₄O₂ (M=246.3 g/mol)

ESI-MS: 247 [M+H]⁺

R_(t) (HPLC): 1.62 min (method K)

Intermediate V.5

5-[(Benzyloxy)methyl]-1H,2H,3H,4H,5H-imidazo[4,5-d]pyridazine-2,4-dione

To a stirred mixture of 29.0 g (118 mmol) intermediate V.4 in 500 mL THFis added 38.2 g (236 mmol) 1,1′-carbonyldiimidazole and the mixture isstirred at reflux for 16 h. After cooling to ambient temperature, theprecipitate is collected by filtration through a sintered funnel. Thecollected solid is washed further with THF and dried under vacuum toyield the desired product.

C₁₃H₁₂N₄O₃ (M=272.3 g/mol)

ESI-MS: 273 [M+H]⁺

R_(t) (HPLC): 1.31 min (method J)

Intermediate V.6

5-[(Benzyloxy)methyl]-1,3-dimethyl-1H,2H,3H,4H,5H-imidazo[4,5-d]pyridazine-2,4-dione

To a stirred solution of 28.0 g (103 mmol) intermediate V.5 in 250 mLDMF is added 42.6 g (309 mmol) potassium carbonate at room temperatureand the mixture is stirred for 30 min. It is then cooled to 0° C. and58.4 g (411 mmol) methyl iodide are added. After warming to ambienttemperature, the reaction mixture is stirred for 16 h. It is quenched byaddition of water and extracted with ethyl acetate (3×). The combinedorganic extracts are washed with cold water to remove DMF, dried overNa₂SO₄ and concentrated. The residue is washed with EtOAc/Petroleumether (1:1) to yield the desired compound.

C₁₅H₁₆N₄O₃ (M=300.3 g/mol)

ESI-MS: 301 [M+H]⁺

R_(t) (HPLC): 1.78 min (method K)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 3.37 (s, 3H), 3.55 (s, 3H), 4.63 (s,2H), 5.52 (s, 2H), 7.25-7.34 (m, 5H), 8.35 (s, 1H).

Intermediate V.7

1,3-Dimethyl-1H,2H,3H,4H,5H-imidazo[4,5-d]pyridazine-2,4-dione

A mixture of 20.0 g (66.6 mmol) intermediate V.6 in 500 mL DCM isprepared and cooled to −78° C. A solution of 58.4 g (499 mmol) borontrichloride in DCM is added and the reaction mixture is stirred at thesame temperature for 2 h. Then, an equimolar mixture of 500 mLMethanol/DCM is added and the reaction mixture is stirred at −78° C. forfurther 2 h. After warming to ambient temperature, the mixture isstirred for another 30 min. The mixture is concentrated andco-evaporated (5 times) with MeOH to remove traces of methyl boronate.The residue is triturated with MTBE and the collected solid is driedunder vacuum to yield the desired compound.

C₇H₈N₄O₂ (M=180.2 g/mol)

ESI-MS: 181 [M+H]⁺

R_(t) (HPLC): 0.75 min (method K)

¹H NMR (500 MHz, DMSO-d₆) δ ppm 3.36 (s, 3H), 3.52 (s, 3H), 8.25 (s,1H), 12.94 (s, 1H).

Intermediate VI

Intermediate VI.1

4,5-Dimethyl 2-bromo-1-methylimidazole-4,5-dicarboxylate

To 30.0 g (114 mmol) 4,5-dimethyl 2-bromo-1H-imidazole-4,5-dicarboxylate(CAS: 705280-65-5) in 300 mL DMF is added 32.4 g (228 mmol) iodomethaneand 31.5 g (228 mmol) potassium carbonate at ambient temperature. Theresulting reaction mixture is stirred for 16 h at ambient temperature.The mixture is then concentrated, diluted with water and extracted withethyl acetate. The organic extract is dried over sodium sulfate andconcentrated. The residue is purified by column chromatography (silicagel, PE/EtOAc 30/70) to yield the desired product.

C₈H₉BrN₂O₄ (M=277.1 g/mol)

ESI-MS: 277/279 [M+H]⁺

R_(t) (HPLC): 1.64 min (method K)

Intermediate VI.2

Methyl 2-bromo-5-formyl-3-methylimidazole-4-carboxylate

A mixture of 20.0 g (72.2 mmol) 4,5-dimethyl2-bromo-1-methylimidazole-4,5-dicarboxylate in 800 mL THF is cooled to−78° C. and 121 mL (180.5 mmol, 25 wt % in toluene) of a solution ofDIBAL-H are added. The reaction mixture is stirred for 2 h and isallowed to warm slowly to −20° C. during this period. The reactionmixture is then quenched with ice cold water and a solution of potassiumsodium tartrate. The mixture is then extracted with ethyl acetate. Thecombined organic extracts are washed with saturated brine, dried oversodium sulfate and concentrated. The residue contains the intermediatealcohol in 85% purity and is used in the next step without furtherpurification.

To a mixture of 15.0 g (85% purity, 51.2 mmol) ethyl2-bromo-5-(hydroxymethyl)-3-methylimidazole-4-carboxylate in 150 mLdichloromethane is added 178 g (2.05 mol) manganese(IV) oxide at ambienttemperature. The resulting suspension is stirred for 16 h at ambienttemperature. The reaction mixture is then filtered through Celite. Thefiltrate is concentrated under reduced pressure to yield the desiredproduct.

C₇H₇BrN₂O₃ (M=247.1 g/mol)

ESI-MS: 247/249 [M+H]⁺

R_(t) (HPLC): 1.51 min (method K)

Intermediate VI.3

2-bromo-3-methyl-5H-imidazo[4,5-d]pyridazin-4-one

To a mixture of 8.0 g (32.4 mmol) methyl2-bromo-5-formyl-3-methylimidazole-4-carboxylate in 80 mL ethanol isadded 1.95 mL (38.9 mmol) hydrazine hydrate at ambient temperature. Thereaction mixture is stirred for 15 min at ambient temperature before 7.4mL (130 mmol) acetic acid is added. The reaction mixture is then heatedto 80° C. and stirred at this temperature for 30 min. After cooling toambient temperature, the precipitated solid is collected by filtration.It is further washed multiple times with ether and dried under vacuum toyield the desired product.

C₆H₅BrN₄O (M=229.0 g/mol)

ESI-MS: 229/231 [M+H]⁺

R_(t) (HPLC): 1.34 min (method K)

Intermediate VI.4

1-Methyl-1H,6H,7H-imidazo[4,5-d]pyridazin-7-one hydrobromide salt

To a mixture of 5.3 g (23.1 mmol)2-bromo-1-methyl-1H,6H,7H-imidazo[4,5-d]pyridazin-7-one in 75 mL MeOH isadded 200 mg Pd/C (10%) in an autoclave reactor. The space above thereaction mixture is evacuated and charged with 50 psi of hydrogenpressure. The reaction mixture is stirred at ambient temperature under50 psi of hydrogen pressure overnight. After removing the hydrogen andbackfilling with nitrogen, the reaction mixture is diluted with 300 mLwarm water and filtered. The filtrate is concentrated to dryness toyield the desired product.

C₆H₆N₄O*HBr (M=231.1 g/mol)

ESI-MS: 151 [M−HBr+H]⁺

R_(t) (HPLC): 0.12 min (method C)

Intermediate VI.5

tert-butyl2-{1-methyl-7-oxo-1H,6H,7H-imidazo[4,5-d]pyridazin-6-yl}acetat

To 2.00 g (8.66 mmol) intermediate VI.4 in 25 mL DMF is added 2.99 g(21.6 mmol) K₂CO3 and the mixture is stirred at RT for 10 min. 1.90 mL(12.98 mmol) tert-butyl bromoacetate are added and stirring at RT iscontinued for 4 h. The reaction mixture is poured onto 50 mL water andextracted with EtOAc. Organic layers are treated with MgSO₄ andcharcoal, filtered, and concentrated under reduced pressure. The crudematerial is stirred with isopropanol, filtered, and the collectedprecipitate is dried at 50° C. to yield the desired product.

C₁₂H₁₆N₄O₃ (M=264.28 g/mol)

ESI-MS: 265 [M+H]⁺

R_(t) (HPLC): 0.82 min (method B)

Intermediate VI.6

2-{1-methyl-7-oxo-1H,6H,7H-imidazo[4,5-d]pyridazin-6-yl}acetic acidhydrochloride salt

To 1.0 g (3.78 mmol) intermediate VI.5 in 10 mL dioxane is added 20 mLHCl in dioxane (4 mol/L) and the reaction mixture is stirred at RT for 3days. The precipitate is filtered off, washed with isopropanol, anddried at 50° C. to yield the desired product.

C₈H₅N₄O₃*HCl (M=244.6 g/mol)

ESI-MS: 209 [M−HCl+H]⁺

R_(t) (HPLC): 0.42 min (method B)

Preparation of Final Compounds Example 1 (General Procedure)5-({3-[(2S)-2-(4-chlorophenyl)-2-hydroxyethyl]-1,2,4-oxadiazol-5-yl}methyl)-1,3-dimethyl-1H,2H,3H,4H,5H-imidazo[4,5-d]pyridazine-2,4-dione

To 200 mg (1.11 mmol) intermediate V.7 in 5 ml DMF is added 380 mg (2.75mmol) K₂CO₃ and the mixture is stirred at RT for 10 min. Subsequently,250 mg (0.92 mmol) intermediate II.1 in 5 mL DMF are added and stirringis continued overnight. The reaction mixture is filtered and thefiltrate purified by reversed phase HPLC (ACN/H₂O gradient, 0.1% TFA) toyield the desired product.

C₁₈H₁₇ClN₆O₄ (M=416.8 g/mol)

ESI-MS: 417 [M+H]⁺

R_(t) (HPLC): 0.86 min (method B)

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 2.92-3.05 (m, 2H), 3.39 (s, 3H), 3.53(s, 3H), 4.92-4.99 (m, 1H), 5.63-5.64 (m, 2H), 7.32-7.37 (m, 4H), 8.40(s, 1H).

The following compounds are prepared using procedures analogous to thosedescribed for example 1 general procedure, using appropriate startingmaterials. As is appreciated by those skilled in the art, theseanalogous examples may involve variations in general reactionconditions.

Starting Reaction Ex. materials Structure conditions 2 V.7 + III.3

1.1 eq int V.7; 3.3 eq. K₂CO₃; DMF, 50° C., 3 h 3 V.7 + III.4

0.9 eq. int. V.7; 1.5 eq K₂CO₃, DMF, RT, over- night 4 V.7 + III.2

1.0 eq. Int. V.7; 2.6 eq K₂CO₃, DMA, RT, 3 h 5 VI.4 + III.1

0.9 eq int. VI.4, 2.8 eq K₂CO₃, DMF, RT, over- night 6 VI.4 + III.3

0.8 eq int. VI.4, 2.9 eq K₂CO₃, DMF, RT, over- night 7 VI.4 + III.5

1.5 eq int. VI.4, 4 eq K₂CO₃, DMF, RT, 4 h 9 VI.4 + III.6

1.0 eq. int. VI.4, 1.9 eq K₂CO₃, DMF, RT, 3 h 10 VI.4 + III.9

1.0 eq. int. VI.4, 1.9 eq K₂CO₃, DMF, RT, 3 d 11 VI.4 + III.8

1.0 eq. int. VI.4, 1.9 eq K₂CO₃, DMF, RT, 3 h 12 VI.4 + III.7

1.0 eq int. VI.4, 1.9 eq K₂CO₃, DMF, RT, 3 d 13 V.7 + III.6

1.0 eq. int. V.7, 1.9 eq K₂CO₃, DMF, RT, 3 h 14 V.7 + III.7

1.0 eq. int. V.7, 1.9 eq K₂CO₃, DMF, RT, 19 h 15 V.7 + III.8

1.0 eq. int. V.7, 1.9 eq K₂CO₃, DMF, RT, 19 h 16 V.7 + III.9

1.0 eq. int. V.7, 1.9 eq K₂CO₃, DMF, RT, 19 h

Analytical data for the compounds described in the table above:

HPLC re- tention time [min] Ex. ESI-MS (method) ¹H NMR (400 MHz,DMSO-d₆) δ ppm  2 397 [M + H]⁺ 0.62 (F) 2.26 (s, 3 H), 2.87-3.03 (m, 2H), 3.39 (s, 3 H), 3.53 (s, 3 H), 4.91 (dd, J = 8.3, 5.3 Hz, 1 H), 5.64(s, 2 H), 7.08-7.13 (m, 2 H), 7.19-7.25 (m, 2 H), 8.39 (s, 1 H)  3 401[M + H]⁺ 0.57 (H) 2.92-3.05 (m, 2 H), 3.39 (s, 3 H), 3.53 (s, 3 H), 4.96(dd, J = 8.2, 5.5 Hz, 1 H), 5.63 (s, 2 H), 7.06-7.14 (m, 2 H), 7.33-7.40(m, 2 H), 8.39 (s, 1 H)  4 423 [M + H]⁺ 0.90 (B) 3.13-3.33 (m, 2 H),3.39 (s, 3 H), 3.52 (s, 3 H), 5.10 (dd, J = 7.9, 5.7 Hz, 1 H), 5.64 (s,2 H), 6.76 (s, 1 H), 7.15-7.30 (m, 2 H), 7.48-7.59 (m, 2 H), 8.36 (s, 1H)  5 387 [M + H]⁺ 0.85 (B) 2.91-3.06 (m, 2 H), 4.05 (s, 3 H), 4.95 (dt,J = 7.9, 5.1 Hz, 1 H), 5.61 (d, J = 4.6 Hz, 1 H), 5.65 (s, 2 H),7.31-7.39 (m, 4 H), 8.40 (s, 1 H), 8.51 (s, 1 H)  6 367 [M + H]⁺ 0.80(B) 2.26 (s, 3 H), 2.85-3.09 (m, 2 H), 4.05 (s, 3 H), 4.91 (dd, J = 8.3,5.3 Hz, 1 H), 5.41 (br s, 1 H), 5.65 (s, 2 H), 7.09 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2 H), 8.40 (s, 1 H), 8.50 (s, 1 H)  7 397 [M +H]⁺ 0.73 (G) 2.87-3.02 (m, 2 H), 4.05 (s, 3 H), 4.83-4.90 (m, 1 H), 5.43(d, J = 4.8 Hz, 1 H), 5.65 (s, 2 H), 5.95-5.97 (m, 2 H), 6.77-6.80 (m, 2H), 6.90-6.93 (m, 1 H), 8.40 (s, 1 H), 8.50 (s, 1 H)  9 427 [M + H]⁺0.48 (A) 3.14-3.18 (m, 2 H), 4.04 (s, 3 H), 5.28 (t, J = 6.7 Hz, 1 H),5.67 (s, 2 H), 7.20 (td, J = 9.0, 2.5 Hz, 1 H), 7.24 (s, 1 H), 7.75 (dd,J = 8.7, 5.3 Hz, 1 H), 7.82 (dd, J = 9.3, 2.3 Hz, 1 H), 8.40 (s, 1 H),8.50 (s, 1 H) 10 411 [M + H]⁺ 0.45 (A) 3.13-3.28 (m, 2 H), 4.04 (s, 3H), 5.09 (dt, J = 7.8, 5.3 Hz, 1 H), 5.66 (s, 2 H), 5.97 (br d, J = 5.6Hz, 1 H) 6.77 (s, 1 H), 7.09 (td, J = 9.2, 2.7 Hz, 1 H), 7.38 (dd, J =8.9, 2.7 Hz, 1 H), 7.54 (dd, J = 8.9, 4.3 Hz, 1 H), 8.39 (s, 1 H), 8.48(s, 1 H) 11 427 [M + H]⁺ 0.49 (A) 3.12-3.28 (m, 2 H), 4.04 (s, 3 H),5.10 (dt, J = 7.9, 5.4 Hz, 1 H), 5.66 (s, 2 H), 5.99 (br d, J = 5.7 Hz,1 H), 6.77 (s, 1 H), 7.28 (dd, J = 8.7, 2.2 Hz, 1 H), 7.56 (d, J = 8.7Hz, 1 H), 7.65 (d, J = 2.2 Hz, 1 H), 8.40 (s, 1 H), 8.48 (s, 1 H) 12 409[M + H]⁺ 0.48 (N) 3.10-3.23 (m, 2 H), 4.05 (s, 3 H), 5.30 (t, J = 6.7Hz, 1 H), 5.68 (s, 2 H), 7.24 (s, 1 H), 7.31 (quind, J = 7.3, 1.5 Hz, 2H), 7.69-7.76 (m, 1 H), 7.86-7.93 (m, 1 H), 8.40 (s, 1 H), 8.50 (s, 1 H)13 457 [M + H]⁺ 0.48 (A) 3.12-3.18 (m, 2 H), 3.39 (s, 3 H), 3.53 (s, 3H), 5.28 (t, J = 6.7 Hz, 1 H), 5.65 (s, 2 H), 7.20 (td, J = 9.0, 2.5 Hz,1 H), 7.24 (s, 1 H), 7.75 (dd, J = 8.7, 5.3 Hz, 1 H), 7.82 (dd, J = 9.4,2.4 Hz, 1 H), 8.39 (s, 1 H) 14 439 [M + H]⁺ 0.47 (A) 3.17 (d, J = 7.1Hz, 2 H), 3.39 (s, 3 H), 3.53 (s, 3 H), 5.30 (t, J = 6.7 Hz, 1 H), 5.66(s, 2 H), 7.24 (s, 1 H), 7.31 (quind, J = 7.3, 1.4 Hz, 2 H), 7.70-7.77(m, 1 H), 7.87-7.93 (m, 1 H), 8.38 (s, 1 H) 15 457 [M + H]⁺ 0.50 (A)3.11-3.28 (m, 2 H), 3.39 (s, 3 H), 3.52 (s, 3 H), 5.06-5.13 (m, 1 H),5.64 (s, 2 H), 5.99 (br s, 1 H), 6.77 (s, 1 H), 7.29 (dd, J = 8.7, 2.1Hz, 1 H), 7.57 (d, J = 8.7 Hz, 1 H), 7.66 (d, J = 1.9 Hz, 1 H), 8.37 (s,1 H) 16 441 [M + H]⁺ 0.45 (A) 3.12-3.28 (m, 2 H), 3.39 (s, 3 H), 3.52(s, 3 H), 5.03-5.14 (m, 1 H), 5.64 (s, 2 H), 5.97 (br d, J = 3.9 Hz, 1H), 6.77 (s, 1 H), 7.09 (td, J = 9.2, 2.7 Hz, 1 H), 7.38 (dd, J = 8.9,2.7 Hz, 1 H), 7.55 (dd, J = 8.9, 4.2 Hz, 1 H), 8.37 (s, 1 H)

Example 86-({3-[(2S)-2-(4-bromophenyl)-2-hydroxyethyl]-1,2,4-oxadiazol-5-yl}methyl)-1-methyl-1H,6H,7H-imidazo[4,5-d]pyridazin-7-one

To 73 mg (0.30 mmol) intermediate VI.6 in 3 ml DMF is added 52 μL (0.30mmol) DIPEA and 58 mg (0.36 mmol) CDI and the mixture is stirred at RTfor 2 min. Subsequently, 78 mg (0.30 mmol) intermediate I1.6 are addedand stirring is continued at 100° C. for 4 h.

After cooling to ambient temperature, the reaction mixture is purifiedby reversed phase HPLC (ACN/H₂O gradient, 0.1% TFA) to yield the desiredproduct.

C₁₇H₁BrlN₆O₃ (M=431.2 g/mol)

ESI-MS: 431/433 [M+H]⁺

R_(t) (HPLC): 0.86 min (method B)

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 2.90-3.05 (m, 2H), 4.05 (s, 3H), 4.94(dd, J=7.8, 5.8 Hz, 1H), 5.65 (s, 2H), 7.26-7.32 (m, 2H), 7.44-7.50 (m,2H), 8.40 (s, 1H), 8.50 (s, 1H)

Analytical HPLC Methods

Method A Vol % water Flow time (mm) (incl. 0.1% TFA) Vol % ACN [mL/min]0.00 99 1 1.6 0.02 99 1 1.6 1.00 0 100 1.6 1.10 0 100 1.6

Analytical column: XBridge BEH C18_2.1×30 mm, 1.7 μm; columntemperature: 60° C.

Method B Vol % water Flow time (mm) (incl. 0.1% TFA) Vol % ACN [mL/min]0.00 97 3 2.2 0.20 97 3 2.2 1.20 0 100 2.2 1.25 0 100 3.0 1.40 0 100 3.0

Analytical column: Stable Bond (Agilent) 1.8 μm; 3.0×30 mm; column temp:60° C.

Method C Vol % water Flow time (mm) (incl. 0.1% TFA) Vol % ACN [mL/min]0.00 97 3 2.2 0.20 97 3 2.2 1.20 0 100 2.2 1.25 0 100 3.0 1.40 0 100 3.0

Analytical column: Sunfire (Waters) 2.5 μm; 3.0×30 mm; columntemperature: 60° C.

Method D Gradient/Solvent Vol % water Flow Time [min] (incl. 0.1% NH₃)Vol % Acetonitrile [ml/min] 0.0 95.0 5.0 1.5 1.3 0.0 100.0 1.5 1.5 0.0100.0 1.5 1.6 95.0 5.0 1.5

Preparative column: XBridge (Waters) C18_3.0×30 mm_2.5 μm; columntemperature: 60° C.

Method E Gradient/Solvent Vol % water Flow Time [min] (incl. 0.1% NH₃)Vol % Acetonitrile [ml/min] 0.0 95.0 5.0 1.5 1.3 0.0 100.0 1.5 1.5 0.0100.0 1.5 1.6 95.0 5.0 1.5

XBridge C18_3.0×30 mm-2.5 μm (Waters); column temperature: 60° C.

Method F Gradient/Solvent Vol % water Vol.-% ACN Flow Time [min] (incl.0.1% TFA) (incl. 0.08% TFA) [ml/min] 0.0 95.0 5.0 1.5 1.3 0.0 100.0 1.51.5 0.0 100.0 1.5 1.6 95.0 5.0 1.5

Preparative column: Sunfire (Waters) C18_3.0×30 mm_2.5 μm; columntemperature: 60° C.

Method G Vol % water Flow time (mm) (incl. 0.1% NH₄OH) Vol % ACN[mL/min] 0.00 97 3 2.2 0.20 97 3 2.2 1.20 0 100 2.2 1.25 0 100 3 1.40 0100 3

Analytical column: XBridge C18 (Waters) 2.5 μm; 3.0×30 mm; columntemperature: 60° C.

Method H Vol % water Vol.-% ACN Flow time (mm) (incl. 0.1% TFA) (incl.0.08% TFA) [mL/min] 0.00 95 5 1.5 1.3 0 100 1.5 1.5 0 100 1.5 1.6 95 51.5

Analytical column: Sunfire C18 (Waters) 2.5 μm; 3.0×30 mm; columntemperature: 60° C.

Method I Gradient/Solvent Vol % water Time [min] (incl. 0.1% TFA) Vol %ACN Flow [ml/min] 0.0 50.0 50.0 1.5 0.02 50.0 50.0 1.5 1.0 0.0 100.0 1.51.1 0.0 100.0 1.5

Analytical column: Sun fire (Waters) CT82× 30 mm_2.5 μm; columntemperature: 60° C.

Method J Gradient/Solvent Vol % water Vol % CAN Time [min] (incl. 0.07%FA) (incl. 0.07% FA) Flow [ml/min] 0.0 97 3 0.6 0.3 97 3 0.6 2.7 2 980.6 3.5 2 98 0.6 3.51 97 3 0.6

Analytical column: AQUITY UPLC BEH C18_2.1×50 mm_1.7 μm; columntemperature: 35° C.

Method K Gradient/Solvent Vol % water Vol % CAN Time [min] (incl. 0.07%FA) (incl. 0.07% FA) Flow [ml/min] 0.0 97 3 0.6 0.3 97 3 0.6 2.2 2 980.6 3.3 2 98 0.6 4.5 2 98 0.6 4.51 97 3 0.6

Analytical column: AQUITY UPLC BEH C18_2.1×50 mm_1.7 μm; columntemperature: 35° C.

Method L Gradient/Solvent Vol % water Vol % CAN Time [min] (incl. 0.1%FA) (incl. 0.1% FA) Flow [ml/min] 0.01 95 5 0.5 4.00 5 95 0.5 5.00 5 950.5 5.20 95 5 0.5 6.00 95 5 0.5

Analytical column: AQUITY UPLC C18_2.1×50 mm_1.8 μm. 100 Å; columntemperature: 25° C.

Method M Gradient/Solvent Vol % water Vol % CAN Time [min] (incl. 0.1%FA) (incl. 0.1% FA) Flow [ml/min] 0.00 95 5 0.5 10.00 5 95 0.5 10.50 595 0.5 11.00 95 5 0.5 12.00 95 5 0.5

Analytical column: AQUITY UPLC C18_2.1×50 mm_1.8 μm. 100 Å; columntemperature: 25° C.

Method N Vol % water Flow Time (mm) (incl. 0.1% TFA) Vol % ACN [mL/min]0.00 99 1 1.6 0.02 99 1 1.6 1.00 0 100 1.6 1.10 0 100 1.6

Analytical column: XBridge Phenyl_2.1×30 mm, 1.7 μm; column temperature:60° C.

The invention claimed is:
 1. A compound according to formula (I):

or a pharmaceutically acceptable salt thereof, wherein: E is:

A is 1,3-benzodioxol-5-yl, phenyl, thiophenyl, benzofuranyl, orbenzothiophenyl, wherein the phenyl, thiophenyl, benzofuranyl, orbenzothiophenyl is unsubstituted or substituted with one or twoindependently selected R¹ substituents; and each R¹ is independentlyhalogen or C₁₋₄ alkyl.
 2. The compound according to claim 1, or apharmaceutically acceptable salt thereof, wherein the compound is apharmaceutically acceptable salt thereof.
 3. The compound according toclaim 2, wherein the pharmaceutically acceptable salt is of a mineralacid or organic acid selected from the group consisting ofbenzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid,fumaric acid, gentisic acid, hydrobromic acid, hydrochloric acid, maleicacid, malic acid, malonic acid, mandelic acid, methanesulfonic acid,4-methylbenzenesulfonic acid, phosphoric acid, salicylic acid, succinicacid, sulfuric acid, and tartaric acid.
 4. The compound according toclaim 1, or a pharmaceutically acceptable salt thereof, wherein A is1,3-benzodioxol-5-yl, phenyl, benzofuran-2-yl, or benzothiophen-2-yl,wherein the phenyl, benzofuran-2-yl, or benzothiophen-2-yl isunsubstituted or substituted with one or two independently selected R¹substituents.
 5. The compound according to claim 1, or apharmaceutically acceptable salt thereof, wherein A is1,3-benzodioxol-5-yl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl,4-methylphenyl, benzofuran-2-yl, 5-fluorobenzofuran-2-yl,5-chlorobenzofuran-2-yl, benzothiophen-2-yl, or6-fluorobenzothiophen-2-yl.
 6. The compound according to claim 1, or apharmaceutically acceptable salt thereof, wherein each R¹ isindependently F, Cl, Br, or CH₃.
 7. The compound according to claim 1,wherein the compound is of the following formula:

or a pharmaceutically acceptable salt thereof.
 8. The compound accordingto claim 1, wherein the compound is of the following formula:

or a pharmaceutically acceptable salt thereof.
 9. The compound accordingto claim 1, wherein the compound is selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition comprising one or more pharmaceutically acceptableexcipients and at least one compound according to claim 1, or apharmaceutically acceptable salt thereof.
 11. A method for inhibitingtransient receptor potential ankyrin 1 activity in a patient, whereinthe method comprises administering to the patient in need thereof atherapeutically effective amount of a compound according to claim 1, ora pharmaceutically acceptable salt thereof.
 12. The method according toclaim 11, wherein the patient has a disease selected from the groupconsisting of cough and idiopathic pulmonary fibrosis (IPF).
 13. Themethod according to claim 12, wherein the patient has idiopathicpulmonary fibrosis (IPF).